Application Of The Pcr-Dgge To Analyze The Community Biodiversity Of Soil Bacteria In Nib8Transgenic Wheat Field

Rhizosphere microbes are the part of crop ecosystem. Interference of the microbial community will cause the epidemic of crop soil-borne diseases. In this experiment, the rhizosphere microbe of transgenic wheat with Nib8gene which was resistant to Wheat Yellow Mosaic Virus (WYMV) was studied. We optimized the traditional microbial DNA extraction method and DGGE electrophoresis conditions.16S rDNA V3variable region of rhizosphere flora were amplified and analyzed with DGGE electrophoresis. The diversity of PCR products was studied by Quantity One.The results showed that SDS high salt method can extract good DNA from Henan rhizosphere microbes, but not from other samples of Luhe, Yangzhou or potted soil; Denaturant plus SDS method was good for total microbial DNA extraction of Luhe samples but not for other regions’ samples; freezing and thawing-Kuni lysozyme lysis method got good DNA from Luhe samples and potted test samples.; phenol/chloroform/isoamyl alcohol extraction method was best one to get the DNA from the four regions’samples based on complete segments, and the brightness of the DNA fragment.It demonstrated that the optimal DGGE denaturant gradient range for the PCR product separation was40%～80%. At low denaturant concentration (0%～40%), the amplified DNA was in double-stranded form and all migrated at the bottom of the gel, while when the denaturant concentration was80%or more, the DNA was totally melted and stayed at the top of the gel. And the optical electrophoresis time was12hours which the PCR products were completely separated.DGGE profiles of LuHe、HeNan、YangZhou and Potted soil were analyzed that before the wheat was sowed the microbial population was the same between the soils of GM wheat and its receptor wheat. At the seedling stage, the difference between transgenic and the receptor was not clear. However between the Greenup and Flowering stage, the microbial population on the rhizosphere of transgenic wheat showed significant differences from the population on the receptor, with the more abundance and the quantity of bacteria. Between the filling stage and maturity, the differences became not obvious.It should be pointed out that Wheat Yellow Mosaic Virus mediated by soil-borne Polymyxa graminis L. was always outbreak at wheat greenup stage. The result of this study showed that the microbial community of GM wheat rhizosphere was more abundant than the normal wheat which implied that it’s the resistant effects of transgenic wheat? or it’s the reason of the wheat resistance? More study is necessary.